Currently, there are over six million adults in the U.S. with myocardial ischemia and more than six hundred human subjects have participated in phase I/II clinical trials to assess the safety of intravascular-administered adenoviral (Ad) vectors to treat cardiovascular diseases. However, such clinical applications are hampered by dose-limiting toxicity stemming from the ability of Ad to potently trigger host innate immune responses. During the previous funding period, researchers in the Nemerow laboratory at The Scripps Research Institute discovered that Ad entry into plasmacytoid dendritic cells induces type one interferon and pro-inflammatory cytokine production via activation of Toll-like receptor nine (TLR9). Thus, these investigators are now in a better position to define the signaling pathways involved in Ad-mediated innate immunity. Moreover, their research efforts uncovered an unanticipated mode of Ad entry into host cells. They demonstrated that partial disassembly of Ad particles in endosomes allows release of an internal capsid protein, designated pVI, which facilitates pH-independent membrane disruption. Thus, these investigators are now in a much better position to extend the knowledge of Ad membrane penetration, a process that is poorly understood for most nonenveloped viruses. In this proposal a combination of biochemical, biophysical, and structural techniques will be used to characterize the membrane lytic properties of pVI as well as its oligomeric structure and precise location and orientation in virus particles. Information arising from these studies will be used as a guide to generate Ad vectors or cell-derived nanoparticles (vaults) containing mutant pVI proteins in order to ascertain the precise role of this capsid protein in endosome disruption. Finally, cell culture and in vivo animal models will be used to investigate the Ad entry processes involved in TLR- dependent and TLR-independent innate immune responses. Together, these studies should significantly increase the knowledge of how a nonenveloped virus penetrates host cells as well as triggers proinflammatory cytokine production. Such information could facilitate the development of safer and more potent viral and non-viral vectors.